Inhibition of murine colorectal cancer metastasis by targeting M2-TAM through STAT3/NF-kB/AKT signaling using macrophage-1 derived extracellular vesicles loaded with oxaliplatin, retinoic acid, and Libidibia ferrea

Abstract

Despite the numerous advances in target therapy for the treatment of colorectal cancer, aggressive colorectal cancer remains an incurable disease whose negative modulation of the immune system in the tumor microenvironment is still critical for improving the patient's prognosis. However, extracellular vesicles (EVs) have demonstrated potential in nanomedicine as drug delivery systems, especially for modulating immune cells in the tumor microenvironment (TME).This study aimed to analyze the combination of oxaliplatin (OXA) with M1-macrophage extracellular vesicles (EV) (M1EV) loaded with retinoic acid (RA) and Libidibia ferrea (LF) to understand its antitumor and anti-metastatic properties in models of Colorectal Cancer (CRC). The cell death profile and the epithelial-mesenchymal transition (EMT) process were analyzed in in vitro models using colorectal cancer cell lines such as CT-26 and MC-38. Macrophage polarization analysis (RAW 264.7 cells) was also performed. Allographic tumors were evaluated by growth curve analysis, weight, qRT-PCR and immunohistochemistry and immunofluorescence. Peritoneal tumors were evaluated by weight, counting the number of tumors found in the peritoneal cavity, histopathological analyses, qRT-PCR and immunohistochemistry. M1EV2 and M1EV3 used alone or particularly M1EV4 negatively regulated tumor progression by TME immunomodulation, leading to a decrease in primary tumor size and metastasis to the peritoneum, liver, and lungs. STAT3, NF-kB and AKT were the main genes downregulated by M1EV systems. Tumor-associated macrophages (TAMs) changed the M2 phenotype (CD163) to the M1 phenotype (CD68), reducing the levels of IL-10, TGF-β and CCL22. Furthermore, malignant cells showed overexpression of FADD, APAF-1, caspase-3 and E-cadherin, and decreased expression of MDR1, survivin, vimentin and PD-L1 after treatment with M1EVs systems. The study shows that M1 anti-tumor macrophage EVs can transport drugs and enhance their immunomodulatory and anti-tumor activity by modulating pathways associated with cell proliferation, migration, survival and drug resistance.